Cell therapy has proven to be highly effective for the treatment of a number of diseases. For example, primary immunodeficiencies can be cured by hematopoetic stem cell transplantation (HSCT) and some leukemia can be brought to complete remission by combined allogeneic HSCT and donor lymphocyte infusion (DLI) (Kolb, H. J. et al., Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 76 (12), 2462-2465 (1990). In some clinical settings, adoptive transfer of virus-specific T cells is very effective to reconstitute immunocompromised patients against life-threatening complications caused by cytomegalovirus (CMV) reactivation (Riddell, S. R. et al., Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 257 (5067), 238-241 (1992)), or lymphoproliferative diseases mediated by Epstein-Ban-Virus (EBV) (Rooney, C. M. et al., Use of gene-modified virus-specific T lymphocytes to control Epstein-Ban-virus-related lymphoproliferation. Lancet 345 (8941), 9-13 (1995)). Similarly, tumor antigen-directed T cells, either derived from autologous tumor-infiltrating lymphocytes or cultured or engineered in vitro, are promising candidates for improved therapies.
Regardless of these interesting clinical observations, a broader transfer of cell therapy to clinical applications is still awaiting. This is mainly due to the fact that most procedures used to generate cell preparations for immunotherapy are very laborious, time consuming and expensive. Furthermore, cell populations known to mediate clinical effects usually need to be enriched to high purities, since contaminating ‘unwanted’ cells can mediate harmful and sometimes life-threatening side effects (like graft-versus-host-diseases (GvHD)-mediating alloreactive T cells upon allogeneic stem cell transplantation and/or DLI treatment). As it has been shown that already very low numbers of adoptively transferred T cells can contribute to beneficial clinical effects, similar rules will also apply for cell populations mediating negative side effects. Therefore, providing high purities of well-defined cell preparations applicable for therapy will become key to make these promising therapies more effective and predictable, as well as to lower the risk of potential side effects.
Current methods for surface marker-mediated clinical cell purification usually rely on single parameters (e.g. CD34, MHC multimers). However, for most cell populations—either directly derived ex vivo or after in vitro cell culture—a combination of different surface markers is necessary in order to truly segregate these cells from others. For example, naturally occurring regulatory T cells (Tregs) represent a promising cell subset, which might be able to prevent acute GvHD upon allogeneic HSCT 4 or the development of autoimmune diseases (Riley, J. L., June, C. H., & Blazar, B. R., Human T regulatory cell therapy: take a billion or so and call me in the morning. Immunity 30 (5), 656-665 (2009), Randolph, D. A. & Fathman, C. G., Cd4+Cd25+ regulatory T cells and their therapeutic potential. Annu Rev Med 57, 381-402 (2006)).
Beside the expression of CD4, which is shared by a large number of cells, additional markers like their constitutive expression of the high-affinity IL-2 receptor a-chain (CD25) are needed to further narrow down heterogeneity. However, CD25 is also expressed on a large fraction of non-regulatory cells, which include recently activated effector and memory T cells. Therefore, combinatorial staining patterns, like combinations of CD4, CD25, CD127, and CD45RA (Miyara, M. et al., Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity 30 (6), 899-911 (2009), Hoffmann, P. et al., Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion. Blood 108 (13), 4260-4267 (2006)) have been suggested to more precisely identify this clinically relevant T cell subset.
All currently available clinical marker-based cell separation techniques utilize paramagnetic beads, which retain labeled cell populations within a magnetic field. Thereby, positive enrichment strategies using directly labeled target populations give highest purities. However, combinations of purifications via several different markers are still difficult to achieve, although being necessary to isolate distinct cell populations of highest purity. In addition, after positive selection, both labels and beads usually remain on the cell product, potentially manipulating the isolated cell population or negatively impacting its functionality/viability e.g. by receptor blockade. Especially in respect to clinical cell sorting, remaining cell labels cause substantial regulatory hurdles for the applicability of cell products into patients. In order to circumvent the problems of positive selection, many clinical cell processing procedures have been changed to depletion settings. Unfortunately, target cell purities are often poor and depletion methods often require a complicated cocktail of different antibodies, which makes their production and application laborsome and expensive.
Thus, there is still a need to provide a method for cell purification or isolation that allows, for example after cell sorting, the release and complete removal of all components of the receptor binding and staining reagents from the purified cell population.